A wide variety of endogenous and exogenous agents can damage the DNA, leading to mutagenesis and genome instability. These, in turn, can lead to human disease, particularly cancer. The mismatch repair system (MMR) is one of multiple DNA repair pathways that recognize and correct DNA damage and is involved in a number of processes to maintain genome integrity. In each process, the crucial first step is recognition of DNA damage by MutS Homolog (MSH) protein complexes, after which the appropriate DNA repair pathway is activated. The long term goal of our research is to understand how recognition of DNA damage is coupled to distinct DNA repair pathways and the way in which MSH proteins are involved in this process. We are particularly interested in the MSH2-MSH3 complex, which recognizes insertion/deletion loops as well as intermediates of genetic recombination. These different substrates are resolved through distinct pathways that require unique downstream factors. We propose that MSH2-MSH3 recognition and binding to specific DNA substrates signals different repair mechanisms. Our research plan will employ a variety of biochemical and genetic approaches to understand the mechanism(s) by which a MSH-DNA interaction triggers the appropriate DNA damage response. In our first Aim, we will characterize MSH2-MSH3 interactions with distinct DNA substrates and examine how MSH2-MSH3 dynamics are affected by that binding. These studies will have broad relevance in understanding how MSH2-MSH3 finds and binds its various substrates to initiate repair. In our second Aim, we will focus on the molecular details of 3' non-homologous tail removal and the role played by MSH2-MSH3. This poorly understood process is critical for single-strand annealing and other double-strand DNA break repair pathways in both yeast and mammalian systems. Our third Aim will focus on the events downstream of MSH2-MSH3-DNA recognition. We will identify and characterize additional proteins that interact with MSH2-MSH3 under differing DNA damaging conditions. In summary, this research will provide important insight into the regulation and coordination of distinct DNA repair pathways, which is critical for maintaining the genetic integrity of a cell and the health of an entire organism.